Mirror image alternative interaction patterns of the same tRNA with either class I arginyl-tRNA synthetase or class II aspartyl-tRNA synthetase

• Published in: Nucleic acids research 1997-12, Vol.25 (24), p.4899-4906
• Main Authors: Sissler, M, Eriani, G, Martin, F, Giege, R, Florentz, C
• Format: Article
• Language: English
• Subjects: Anticodon - chemistry; anticodon loop; arginine; arginine-trna ligase; Arginine-tRNA Ligase - classification; Arginine-tRNA Ligase - metabolism; aspartate-trna ligase; Aspartate-tRNA Ligase - classification; Aspartate-tRNA Ligase - metabolism; aspartic acid; Base Sequence; binding; Biochemistry, Molecular Biology; cloverleaf structure; complexed transfer rna; crystal structure; crystals; DNA Footprinting; Escherichia coli; Fungal Proteins - classification; Fungal Proteins - metabolism; interactions; Life Sciences; ligases; Models, Molecular; molecular conformation; Molecular Sequence Data; Nucleic Acid Conformation; nucleotide sequences; Protein Binding; Recombinant Fusion Proteins - metabolism; RNA, Fungal - chemistry; RNA, Fungal - metabolism; RNA, Transfer, Arg - chemistry; RNA, Transfer, Arg - metabolism; RNA, Transfer, Asp - chemistry; RNA, Transfer, Asp - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Stereoisomerism; Substrate Specificity; transfer RNA
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/25.24.4899

Gene cloning, overproduction and an efficient purification protocol of yeast arginyl-tRNA synthetase (ArgRS) as well as the interaction patterns of this protein with cognate tRNAArg and non-cognate tRNAAsp are described. This work was motivated by the fact that the in vitro transcript of tRNAAsp is of dual aminoacylation specificity and is not only aspartylated but also efficiently arginylated. The crystal structure of the complex between class II aspartyl-tRNA synthetase (AspRS) and tRNAAsp, as well as early biochemical data, have shown that tRNAAsp is recognized by its variable region side. Here we show by footprinting with enzymatic and chemical probes that transcribed tRNA-Asp is contacted by class I ArgRS along the opposite D arm side, as is homologous tRNAArg, but with idiosyncratic interaction patterns. Besides protection, footprints also show enhanced accessibility of the tRNAs to the structural probes, indicative of conformational changes in the complexed tRNAs. These different patterns are interpreted in relation to the alternative arginine identity sets found in the anticodon loops of tRNAArg and tRNAAsp. The mirror image alternative interaction patterns of unmodified tRNAAsp with either class I ArgRS or class II AspRS, accounting for the dual identity of this tRNA, are discussed in relation to the class defining features of the synthetases. This study indicates that complex formation between unmodified tRNAAsp and either ArgRS and AspRS is solely governed by the proteins.